The overall objective of this proposal is to understand the function of enamel gene products (i.e., amelogenins, enamelins and enamel proteinases), to advance the understanding of the process of amelogenesis, to create a strategy to produce an enamel bioceramic as a novel dental restorative biomaterial, and to improve the diagnosis of the various types of amelogenesis imperfecta. We hypothesize the motifs within the anionic enamelin protein (e.g.tuftelin) structure serve to nucleate the initial crystalline phase, and that motifs within amelogenin protein aggregates regulate the size, orientation and patterns of the growing enamel crystals. We further predict that proteases (e.g. calcium-dependent metalloproteinases) degrade proteins and facilitate the removal of water and protein from the mineralizing extracellular matrix. To address this hypothesis, the following Specific Aims have been formulated: (1) To determine the function of amelogenins and enamelins during mouse amelogenesis in in vitro ameloblast cell culture, in transfected odontoblast cell cultures and mouse molar tooth organ culture systems using antisense inhibition strategies. (2) To determine the function of enamelins to nucleate octacalcium phosphate (OCP) or hydroxyapatite (HAP) in vitro. Recombinant enamelins will be evaluated as nucleators for OCP or HAP in vitro. (3) To determine the function of amelogenins to regulate the size and patterns of OCP or HAP crystal growth in vitro. Recombinant amelogenins will be evaluated as regulators for the crystal growth and patterns of growth in vitro. Subsequent protein engineering approaches using site-directed mutagenesis will enable the identification of the motifs required for OCP or HAP crystal growth along the c-axis as well as the patterns of crystal growth. (4) To determine the function of enamel proteases to regulate both structural properties and the stepwise removal of enamel proteins and subsequently to regulate crystal formation. We propose to isolate and characterize the enamel protease gene product. (5) To fabricate enamel HAP crystal formation in vitro. Enamel replacement therapy is a strategy to use recombinant enamel protein motifs to fabricate an enamel bioceramic as a possible dental enamel restorative biomaterial. These experiments are a logical extension of the proposed functional studies to define how enamel proteins regulate enamel OCP and HAP crystal formation.